For understanding specific properties and functions in various types of biopolymers such as protein and complexes thereof, detailed steric structures thereof are indispensable information. From the basic biochemical viewpoint, for example, information on the three-dimensional structure of protein or the like serves as the basis for understanding the mechanism of function appearance in a biochemical system by an enzyme or hormone. Particularly in the fields of pharmaceutical science, genetic engineering and chemical engineering among industrial circles, the three-dimensional structure provides information indispensable for rational molecular design for facilitating drug design, protein engineering, biochemical synthesis and the like.
As a method of obtaining three-dimensional steric structural information of such biopolymers at atomic levels, X-ray crystal structural analysis is the most cogent and high-accuracy means at present. Analytic speeds are remarkably improving by rapid improvement of arithmetic processing speeds of computers in addition to reduction of measuring times and improvement of measuring accuracy due to recent hardware improvement of X-ray light sources-analyzers, and the three-dimensional structures are conceivably going to be clarified with the main stream of the X-ray crystallographic analysis also from now on.
In order to decide the three-dimensional structure of a biopolymer by X-ray crystal structural analysis, on the other hand, it is indispensable to crystallize the target substance after extraction-purification. At present, however, there is neither technique nor apparatus which can necessarily crystallize any substance when applied, and hence crystallization is progressed while repeating trial and error drawing on intuition and experience under the present circumstances. A search by an enormous number of experimental conditions is necessary for obtaining a crystal of a biopolymer, and crystal growth forms the main bottleneck in the field of the X-ray crystallographic analysis.
Crystallization of a biopolymer such as protein is basically adapted to perform a treatment of eliminating a solvent from water or an anhydrous solution containing the polymer thereby attaining a supersaturated state and growing a crystal, similarly to the case of a general low molecular weight compound such as inorganic salt. As typical methods therefor, there are (1) a batch method, (2) dialysis and (3) a gas-liquid correlation diffusion method, which are chosen in response to the type, the quantity, the properties etc. of a sample.
The batch method is a method of directly adding a precipitant eliminating hydration water to a solution containing a biopolymer for reducing the solubility of the biopolymer and converting the same to a solid phase. In this method, solid ammonium sulfate, for example, is frequently used. This method has such disadvantages that the same requires a large quantity of solution sample, fine adjustment of a salt concentration and pH is difficult, skill is required for the operation, and reproducibility is low. As shown in FIG. 45, for example, the dialysis, which is overcoming the disadvantages of the batch method, is a method of sealing a solution 52 containing a biopolymer in the interior of a dialytic tube 51 for continuously changing the pH etc. of a dialytic tube outer liquid 53 (e.g., a buffer solution) and making crystallization. According to this method, the salt concentrations of the inner and outer liquids and the pH difference are adjustable at arbitrary speeds, and hence the conditions for crystallization are easy to find out. As shown in FIG. 46, for example, the gas-liquid correlation diffusion method is a technique of placing a droplet 62 of a sample solution on a sample holder 61 such as a cover glass and placing this droplet and a precipitant solution 64 in a sealed container 63, thereby slowly setting up an equilibrium by evaporation of volatile components therebetween.
However, there are various problems in crystallization of a biopolymer such as protein, as described above in the present circumstances. First, the crystallinity is not excellent. A biopolymer contains a large quantity of solvent (mainly water) (.gtoreq.50 volume %), dissimilarly to crystals of other substances. This solvent is disorderly and readily movable in the intermolecular clearances of the crystal. Although the molecules are gigantic, further, there is substantially no wide-ranging intermolecular packing contact in the crystal, and only slight molecule-to-molecule contact or contact by hydrogen bond through water molecules is present. The crystallinity is not excellent due to such factors. Second, it is extremely sensitive to crystal conditions. While the biopolymer is stabilized in the solvent by interaction between individual molecular surfaces, charge distributions on the molecular surfaces, particularly conformation of amino acids in the vicinity of the molecular surfaces etc., extremely vary with the environment, i.e., the pH, the ionic strength and the temperature of the solution, the type and the dielectric constant of the buffer solution and the like. Therefore, the crystallization process becomes a multi-parameter process in which complicated various conditions are entangled with each other, and it has been impossible to establish a unific technique which is applicable to any substance. As to protein, crystallization of hydrophobic membrane protein is extremely difficult at present although it is biochemically extremely important as compared with water-soluble protein, and only two examples have heretofore performed crystallization and further succeeded in analysis of high resolution.
As described above, crystallization of biopolymers such as protein and complexes thereof forms the most significant bottleneck for the X-ray crystal structural analysis since the same has heretofore been progressed while repeating trial and error, although this is an important process in science and industry. Therefore, it is necessary to hereafter understand the basic principle of crystallization and develop a crystallization technique which is applicable to any molecule.